EXPERIMENTAL INFECTION OF LAYING HENS WITH SALMONELLA ENTERICA SEROVAR GALLINARUM

Brazilian Journal of Microbiology (2005) 36:51-56 ISSN 1517-8382 EXPERIMENTAL INFECTION OF LAYING HENS WITH SALMONELLA ENTERICA SEROVAR GALLINARUM Gláucia Helaine de Oliveira; Angelo Berchieri Junior*; Alexandre César Fernandes Departamento de Patologia Animal, Faculdade de Ciências Agrárias e Veterinárias da Universidade Estadual Paulista, Jaboticabal, SP, Brasil Submitted: April 15, 2003; Returned to authors for corrections: July 03, 2004; Approved: September 27, 2004 ABSTRACT Experimental infections were set up in commercial laying birds, comprising a white relatively resistant line and a red susceptible line infecting with Salmonella enterica serovar Gallinarum. The major findings were that in susceptible birds clinical disease occurred in a dose-dependent manner. Faecal excretion occurred in susceptible birds almost up to death but also occurred in the more resistant line and in birds, which were convalescing. Removal of birds, which had died from the disease, from the environment, reduced the resultant mortality/ morbidity and may be regarded as a useful measure for control. Key words: laying hens, Salmonella Gallinarum, contact infection INTRODUCTION The disease induced by infection with Salmonella enterica serovar Gallinarum in chickens is usually observed in adult birds, although the etiologic agent is very pathogenic for chickens of all ages (1,8). There is considerable information available on the biology of the microorganism and some aspects of its relationship with the host, although this is relatively elementary in nature (8,10,12). The disease remains an important economic problem and its control has been attempted using vaccination with a live attenuated strain of Salmonella Gallinarum or by chemotherapy. Both of these are not, however, completely satisfactory since the live vaccine retains some virulence (6,13) and many drugs are prohibited for use in animal production. Besides, drug therapy is expensive and sometimes increases mortality because of toxicity resulting from misuse (3). Another possibility for the future is the replacement of susceptible varieties of birds by others that are more resistant to clinical disease, as the light white varieties usually are (4). Light white varieties of chickens may not exhibit typical symptoms of fowl typhoid but can show sub-clinical disease with the bacterium persisting in the bird longer than it does in susceptible lines (1). The present study was carried out to define some aspects of the progress of the infection of Salmonella Gallinarum in commercial laying hens. In the white variety which is often resistant to the clinical disease it was assessed the persistence of the bacterium despite of the absence of signals of fowl typhoid and in the brown birds it was assessed the transmission by feces, by eggs, and by contact between infected and non-infected birds. MATERIALS AND METHODS The method for enumeration of Salmonella Gallinarum was that adopted by Berchieri Jr. et al. (1). Briefly, an overnight broth culture of Salmonella Gallinarum strain 9, resistant to nalidixic acid (SGNal r ) was prepared and incubated in a shaking (100 strokes/min) incubator at 37ºC. In experiment one, 48 newly-hatched chickens from a light white variety of Hy-Line were separated into two groups. In one group (group B) the birds were inoculated orally with 0.1 ml of the neat culture containing approximately 8.5 x 10 8 colony forming units/ml (cfu/ml) and in the other group (group A) the culture was diluted by a factor of 10-3, thereby containing approximately 8.5 x 10 5 cfu/ml. The 5 days old birds were inoculated. After 3, 7, 14, 21, 28 and 35 days post-inoculation *Corresponding Author. Mailing address: Departamento de Patologia Animal, Faculdade de Ciências Agrárias e Veterinárias, UNESP. Via de Acesso Prof. Paulo Donato Castellane, s/n. 14884-900, Jaboticabal, SP, Brasil. E-mail: berchier@fcav.unesp.br 51

G.H. de Oliveira et al. (dpi), birds were killed for bacteriological examination of the liver, spleen, ovary, blood and caecal contents. The samples were diluted in PBS (1:10) and homogenised and further diluted in a decimal series. The dilutions were plated on Brilliant green agar (Oxoid CM265) containing nalidixic acid (25 μg/ml) and novobiocin (2 μg/ml) (BGNNov). The plates were incubated overnight at 42ºC and the cfu/ml were transformed to log 10 for analysis. In the absence of bacterial growth the sample was cultured in selenite broth (Oxoid.CM395) containing novobiocin (40 mg/l) overnight at 42ºC and then plated on BGNNov, which was incubated at 42ºC overnight. In addition, two groups of day-old commercial brown egg laying variety of Hy-Line were infected in the same way to observe morbidity and mortality. A second experiment was carried out with 48 adult (18 weeks old) laying hens from a brown egg-layer flock (Hy line) placed in two groups. The birds in group B were inoculated orally with 1 ml of a neat culture containing approximately 8.5 x 10 8 cfu/ml and in the other (group A) 1 ml of the culture diluted to 10-3, thereby containing approximately 8.5 x 10 5 cfu/ml. Birds from both groups were killed after 3, 7, 14, 21, 28 and 35 dpi for bacteriological examination as described above. At this time, eggs laid were also examined. They were collected in a sterile glass jar, broken by agitation and incubated overnight at 42ºC. From the jar, a cotton swab was used to collect a sample of egg, which was streaked out on BGNNov and incubated overnight at 42ºC. This experiment was repeated later with four groups of commercial brown egg laying hens, using four dilutions of the inoculum (neat: culture containing approximately 8.5 x 10 8 cfu/ ml; 10-1 : 8.5 x 10 7 cfu/ml; 10-2 : 8.5 x 10 6 cfu/ml and 10-3 : 8.5 x 10 5 cfu/ml) for bacteriological examination of the faecal excretion and eggs laid. A third experiment was done with 190 adult (18 weeks old) commercial brown egg laying hens (Hy line). The birds were separated in group A and group B. They were inoculated orally with 1 ml of an overnight broth culture containing approximately 8.5 x 10 8 cfu/ml of Salmonella Gallinarum. They were placed together with uninfected birds. In group A dead birds were taken out as soon as they were noticed and in group B dead birds were left remaining in the cage for 48 hours. Cloacal swabbing were taken twice a week and all eggs laid were done through out the experiment. Data from the two groups were compared using the χ 2 test of significance (14) Two additional experiments were done to assess faecal excretion and egg contamination. In one of them birds from a new variety of brown egg Brazilian line were infected with neat or diluted SGNal r cultures. In the second additional experiment, the 40 adult (18 weeeks old) brown varieties of chickens (Hy line) were tested again, but using four doses of inoculum containing undiluted (8.5 x 10 8 cfu/ml), 10-1 (8.5 x 10 7 cfu/ml), 10-2 (8.5 x 10 6 cfu/ml) and 10-3 (8.5 x 10 5 cfu/ml) diluted cultures. RESULTS In all experiments cloacal swab and serological examination of the birds carried out at the beginning of each experiment did not show any evidence of Salmonella. During experiment one, mortality was not observed among the variety of young light birds and Salmonella Gallinarum was not recovered from caecal or cloacal samples. Rates of organ infection were higher among birds that received the undiluted culture. However, in both groups Salmonella Gallinarum (SG) was found in liver and/or spleen 35 days post-infection (Table 1). Table 1. Salmonella Gallinarum detected in samples of tissue of the light variety of laying hens. Number (log 10 ) of viable cells per gram of the sample after any interval of time after infection 3 days 7days 14 days 21 days 28 days 35 days Bird nº L S L S Ov L S Ov B L S B L S L S 1 - - - - - 2.78 3.40 - - - - - - - - - 2 - - - - - + 2.60 2.90 - + + 1.70 - - 1.48 + 3 - - - - - - - - - + 3.00 - - - - - 4 - - - - - + + - - - - - + 2.48 - - 5 2.36 2.40 2.00 2.48 2.70 + + + - + - - - - - + 6 + 2.08 2.60 4.04 + - - - - + + - - - - + 7 2.48-2.48 3.52 2.95-6.45 - - - + - + 2.00 - + 8 2.00 + 2.26 3.30-2.30 3.00 3.30 + + 2.00 - - - - - L: liver, S: spleen, Ov: ovary, B: blood. Negative results from the blood, ovary and caecal contents examination were excluded from the table; Group B: birds number 1 to 4: inoculated with 0.1 ml of an overnight broth culture diluted at 10-3 (8.5 x 10 5 cfu/ml); Group A: birds number 5 to 8: inoculated with 0.1 ml of a net (8.5 x 10 8 cfu/ml) overnight broth culture; +/-: positive/negative after incubation in selenite broth. 52

Infection of laying hens with S. Gallinarum Mortality was high in young red birds that received the neat culture of SG (16 died of 17 infected), whereas only one red bird died (1/17) in the group challenged with the broth culture diluted to 1:1000. At the end of the experiment, bacteriological examination failed to isolated SG from the liver or spleen samples of any of the surviving red birds. Table 2 shows the results of bacteriological examination of adults birds of the commercial variety of brown egg line. Inspection was carried out up to 35 days post infection (dpi). However, 21 dpi only two birds remained in the flock inoculated with the higher dose and in the flock inoculated with the smaller dose three birds were examined at 28 and 35 dpi. As before, negative results related to samples collected 14, 21, 28, and 35 dpi are not shown in table two. The mortality among adult red birds was comparable with that seen with young red birds. SG was isolated in birds that received the lower dose in the first two weeks after infection but not thereafter. Those birds from which SG was countable in 14 and 21 dpi were clinically close to death. Mortality was high in the other group. No infected eggs were found during the experiment. The third experiment (Table 3) was done to assess infection by contact between experimentally infected and uninfected birds. When dead birds were removed from the cage as soon as they were noticed the disease did not spread (7/110=6%) as it did when dead birds were allowed to remain in the cage (40/83=48%) indicating the importance of infection from infected dead animals, possibly through cannibalism. This is statistically highly significant (χ 2 = 44.95, P< 0.01). In this experiment, cloacal swabs were taken twice a week and all 186 eggs were examined. No Table 3. Salmonella Gallinarum Infection by contact. Group* A Group B Experiment D/I** D/C*** D/I D/C 01 4/6 1/6 02 7/10 0/30 03 5/6 0/6 4/5 4/5 04 5/5 0/10 4/5 7/10 05 5/6 0/22 11/12 17/40 06 8/22 6/36 7/20 12/36 Total 34/55 7/110 26/42 40/83 *Group A: dead birds were taken out as soon as noticed; Group B: dead birds remained in cage for 48 hours; **Number of dead birds/number of infected birds; *** Number of dead birds/number of contact birds. Salmonella-contaminated eggs were found. Salmonella was recovered by cloacal swab just before death in 16 birds. However, some birds presented a different behaviour, especially those that did not die. These birds excreted SGNal r in the faeces for several days (Table 4). Two additional experiments were done to assess faecal excretion and egg contamination. In one of them birds from a new variety of brown egg Brazilian line were infected with neat or diluted SGNal r cultures. These birds were very resistant to infection with minimum mortality, and the birds excreted SGNal r similarly to the white variety (data not shown). In the second Table 2. Salmonella Gallinarum isolated from organs and caecal contents of a commercial brown egg adult laying hens Viable number (log 10) of Salmonella organisms per gram of the sample after any interval of time after infection 3 days 7 days 14 days 21 days Bird nº L S Ov B C L S Ov B C L S Ov C S Ov 1 - - - - - 4.62 4.08 + 2.08 - - - - - - - 2 - - - - - - - - - - 3.20 4.08 + 4.45 - - 3 - - - - - - - - - - - - - - 2.78 + 4 - - - - - - - - - - - - - - - - 5 4.04 - - 2.00-4.56 3.36 + - - - - + - - - 6 4.20 4.08 2.00 - - 8.08 8.08 + 4.65 3.60 2.60 4.08 - - - - 7 - - - - - 2.00 3.26 + - - - - - - nd nd 8 2.60 3.95 + - 5.00 4.30 3.17 + - + 3.51 2.08 + - nd nd L: liver, S: spleen, Ov: ovary, B: blood, C: caecal contents; Group B: birds number 1 to 4: inoculated with 1 ml of an overnight broth culture diluted at 10-3 (8.5 x 10 5 cfu/ml); Group A: birds number 5 to 8: inoculated with 1 ml of a net (8.5 x 10 8 cfu/ml) overnight broth culture; +/-: positive/negative after incubation in selenite broth; nd: not done because bird died before 53

G.H. de Oliveira et al. Table 4. Salmonella Gallinarum 9Nal r isolated by cloacal swab from adult red laying hens. Days post-infection Bird nº 7** 8 9 10 12 13 20 21 22 23 25 26 65 I* +(9) 53 I +(10) 31 I +(10) 21 I +(12) 25 I + +(13) 44 C + 87 I +(10) 61 I +(14) 94 C +(21) 54 C +(21) 78 I +(21) 60 C + + + 72 C +(24) 60 C 72 C 82 C + 98 C + + 27 28 29 30 31 32 33 34 36 44 45 60 C + + + + 82 C + + + 98 C + +(30) 42 C + 85 C +(32) 74 C +(32) 95 C + +(34) 99 C + +(35) 36 C + + The swabs were daily after infection. *I: Infected bird, C: Birds that acquired the infection by contact; ** +: positive cloacal swab. Number in parenthesis indicates the day that birds died; NB: 46 66 dpi, cloacal swabs from all birds were negative. additional experiment, the adult brown variety of chickens were tested again, but using four doses of inoculum containing undiluted 10-1, 10-2 and 10-3 diluted cultures. As observed previously, Salmonella was isolated from the faeces almost to the point of death, while some of those that survived did excrete the bacteria for some days (Table 5). Salmonella Gallinarum was not recovered from any of 460 eggs laid. DISCUSSION AND CONCLUSION Previous work has shown that adult birds of light white varieties, which are considered from field data to be more resistant to fowl typhoid, survived the experimental infection. Nevertheless, some birds had transitory bacteraemia (1). We have also found that age is not a limiting factor for disease occurrence such was shown before (11,12). In the two groups of day-old red birds included in this experiment, mortality was also high among those inoculated with the neat culture and sub-clinical disease was seen among those that received the low dose as happened with the adult red birds. In the experiment with adult red birds, the green/green yellowish diarrhoea started 2-4 days after infection, but the birds continued eating, drinking and laying. Strong changes occurred closer to death. Birds stopped eating, drinking and laying. At this point, Salmonella Gallinarum spread out rapidly through the population of adult birds, being present in the carcass and in the cloacae. These results suggest that the propensity towards disease-free carriage and the production of contaminated egg are less marked in susceptible birds, what agrees with previous studies (1,2), although similarities occur with the epidemiology of pullorum disease in different lines (10). In those birds that received the low dose no mortality occurred. Thus, when birds are susceptible, the occurrence of disease may be dose dependent as found previously (1) and birds that survived, whatever the dose is, did not necessarily become carriers as occurred with birds infected by Salmonella Pullorum (1,7). Faecal excretion was eventually seen among varieties of birds considered resistant to Salmonella Gallinarum (2). In the present study, in the group infected with the low dose only one bird died (Table 2). Salmonella Gallinarum was not recovered from cloacae and eggs (1). However, in the experiment to assess contact infection (Tables 3 and 4), although frequent faecal excretion of Salmonella Gallinarum was not observed as occurred in Salmonella Enteritidis infection (5), some birds that did not die did excrete the bacterium for several days. Unlike previous results (1), these findings demonstrate that, among susceptible birds, survivors may contain and excrete Salmonella. Thus, faecal excretion is not related to the variety of the birds. 54

Infection of laying hens with S. Gallinarum Table 5. Salmonella Gallinarum 9Nal r isolated by cloacal swab from adult red laying hens. Days post-infection Bird nº 24h 48h 3 5 7 9 11 13 15 17 19 21 23 25 27 1 + (11) 2 + (8) 3 + (8) 4 + (10) 5 (9) 6 (15) 7 (8) 8 + (19) 9 + (11) 10 + + 11 (11) 12 + 13 + + (28) 14 (8) 15 (7) 16 (10) 17 + (9) 18 (16) 19 + (10) 20 + (9) 21 + + 22 + + 23 + (20) 24 (10) 25 + + + + 26 + +(28) 27 28 29 (8) 30 + 31 + 32 33 (11) 34 35 36 37 + (12) 38 39 40 (13) * Birds 1-10: receiving neat culture (8.5 x 10 8 cfu/ml), Birds 11-20: receiving culture diluted to 10-1 (8.5 x 10 7 cfu/ml), Birds 21-30: at 10-2 (8.5 x 10 6 cfu/ml), and birds 31-40: at 10-3 (8.5 x 10 5 cfu/ml). Number in parenthesis indicates the day that birds died. In conclusion, the work have shown that 1.Salmonella Gallinarum was isolated from internal organs of birds with no disease up to 35 days as well as in feces; 2.The disease is dose dependant to the susceptible variety; 3.Eggs laid might not be contaminated by SG and the dissemination of the disease is strongly related with the presence of dead bird in the cage. ACKNOWLEDGEMENTS The authors are grateful to FAPESP and CNPq for financial support and to A. J. dos Santos and A. R. Batista for their technical assistance. RESUMO Infecção experimental com Salmonella enterica serovar Gallinarum em poedeiras comerciais Infecções experimentais por Salmonella enterica serovar Gallinarum foram realizadas em aves de postura comercial, incluindo uma linhagem branca resistente e uma linhagem vermelha susceptível ao desenvolvimento da enfermidade clínica. As aves de linhagem susceptível apresentaram doença clínica dependente da dose administrada. Excreção fecal foi observada em aves da linhagem susceptível próximo ao momento da morte e, eventualmente, em aves da linhagem resistente e aves convalescentes. A remoção das aves mortas do meio ambiente reduziu a taxa de mortalidade/morbidade, procedimento este que pode ser utilizado como medida de controle. Palavras-chave: ave de postura comercial; Salmonella Gallinarum; infecção por contato REFERENCES The third experiment showed that by removing dead birds quickly, fowl typhoid might be prevented. In addition, the data from this work strongly suggest that the basic principles of hygiene and cleaning are very important in the prevention of fowl typhoid, possibly more so than the use of any kind of therapy (9). 1. Berchieri Jr., A.; Murphy, C.K.; Marston, K.; Barrow, P.A. Observation on the persistence and vertical transmission of Salmonella enterica serovars Pullorum and Gallinarum in chickens: effect of bacterial and host genetic background. Avian Path., 30, 221-223, 2001. 2. Berchieri Jr., A; Oliveira, G.H.; Pinheiro, L.A.P.; Barrow, P.A. Experimental Salmonella Gallinarum infection in light laying hens lines. Braz. J. Microbiol., 31, 50-52, 2000. 55

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